CN101871018A - Real-time fluorescent RT-PCR method for detecting whether arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus infect host - Google Patents
Real-time fluorescent RT-PCR method for detecting whether arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus infect host Download PDFInfo
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Abstract
The invention discloses a real-time fluorescent RT-PCR method for detecting whether arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus infect a host. In the invention, according to the RNAs of the arabis mosaic virus, the bean yellow mosaic virus and the lily symptomless virus, a specific amplimer and a fluorescent probe are designed, RT-Realt time PCR reaction is carried out the RNA template of virus to be identified by means of the specific amplimer and the fluorescent probe, and whether the arabis mosaic virus, the bean yellow the mosaic virus and the lily symptomless virus infect the host is rapidly detected by carrying out real-time fluorescent detection on RT-Real time PCR products.
Description
Technical field
The present invention relates to viral species detection technique, be specifically related to the Fast Detection Technique whether arabis mosaic virus, bean virus 2 and lily asymptomatic virus infect the host.
Background technology
Arabis mosaic virus (Arabis mosaic virus, ArMV), bean virus 2 (Bean yellow mosaic virus, BYMV) and lily asymptomatic virus (Lily symptomless virus, LSV) three kinds of viruses are to infect the main diseases original of lily and gladiolus, bring bigger financial loss to the grower.The arabis mosaic virus host range is extensive, can infect about 174 and belong to 215 kind of plant, natural infection also endangers multiple flowers, melon, beans and fruit tree, causes symptoms such as floral leaf, yellow chlorisis, dwarfing, shrinkage even necrosis, is the inward Plant Quarantine harmful organism of China's regulation.The BYMV virus host is extensive, and main host plants is ornamental plant and leguminous plantss such as gladiolus.The gladiolus growth of seedling gesture that infected by BYMV weakens, and white broken spot appears in blade, spends variegated, cause the cut-flower downgrade, plant ball output and reduce, and the broad bean that infected by BYMV, plant biography rate 4%～17%, the field sickness rate can cause 59%～96% production loss up to 67%～100%.Lily is subjected to the infection rate of LSV up to 73%, one non-evident sympton when it infects separately, and normal under field conditions (factors) and compound the infecting of other virus causes serious floral leaf of lily blade and whole strain to be downgraded, and influences output and the commercial value of lily.
Arabis mosaic virus, lily asymptomatic virus and bean virus 2 detect and can adopt conventional enzyme-linked immunosorbent assay method (ELISA) to detect, but this detection method sensitivity is low, has deviation; Particularly when gladiolus and lily are in dormant state, it is lower to plant in the ball virus concentration, adopts conventional enzyme-linked immunosorbent assay method (ELISA) then can not detect.Deng Cong is good to be waited according to the different isolate coat protein gene conservative of ArMV sequence, design specific detection primer and probe, set up the real-time RT-PCR detection method that detects ArMV, for example Chinese patent CN1952649A has effectively solved the low technological deficiency of enzyme-linked immunosorbent assay method (ELISA) sensitivity.
When the staff utilizes the real-time RT-PCR detection technique that the three kinds of main diseases originals (arabis mosaic virus, bean virus 2 and lily asymptomatic virus) that infect lily and gladiolus are detected, need do test experience three times, speed is slow, therefore needs to improve.
Summary of the invention
To be solved by this invention is the slow technical problem of detection speed whether arabis mosaic virus, bean virus 2 and lily asymptomatic virus infect the host.
Address the above problem and adopt following technical scheme: whether arabis mosaic virus, bean virus 2 and lily asymptomatic virus infect host's real-time fluorescent RT-PCR method for detecting, comprise the steps:
A, go up total RNA that extracting goes out virus to be detected from the host;
B, total RNA is diluted to RNA solution;
C, with RNA solution as template, carry out RT-Real time PCR reaction; In RT-Real time PCR reaction system, comprise amplimer, fluorescent probe; Wherein amplimer is totally three groups, is respectively primer ArMV, primer BYMV and primer LSV, has following sequence respectively:
Upstream primer ArMV-F:5 '-TTTATGCGATCTCGGGACCTA-3 ',
Downstream primer ArMV-R:5 '-TTGCACAACCACTGGCATCT-3 ',
Upstream primer BYMV-F:5 '-GGAGCAGGTGACATACCCTTTAA-3 ',
Downstream primer BYMV-R:5 '-TCCGCAACTTCCGAGAAATG-3 ',
Upstream primer LSV-F:5 '-GCAGCATTGAGTTTGAGAATGG-3 ',
Downstream primer LSV-R:5 '-GTCCAGCGTGCTTCTTCATG-3 ',
Primer ArMV, primer BYMV and primer LSV be respectively according to the high conservative capsid protein gene sequences Design of arabis mosaic virus, bean virus 2 and lily asymptomatic virus, and do not do mutually between primer,
Wherein fluorescent probe is totally three kinds, is respectively probe ArMV-P, probe BYMV-P and probe LSV-P; Wherein
Probe ArMV-P is the sequence that 5 ' end 5-Fluoresceincarboxylic acid is modified, 3 ' end 5-carboxyl tetramethyl-rhodamine is modified: 5 '-FAM-TGCCCCAAGTGGAGAAACTGCA-TAMRA-3 ', when probe and target sequence pairing, FAM fluorophor emitted fluorescence is because of approaching by cancellation with the TAMRA quencher of 3 ' end, when carrying out extension, 5 ' 5 prime excision enzyme activity of polysaccharase cuts off probe, make the FAM fluorophor separate emitting fluorescence with quencher TAMRA
Probe BYMV-P is the sequence that 5 ' end chlordene-6-methyl fluorescein is modified, 3 ' end 5-carboxyl tetramethyl-rhodamine is modified: 5 '-HEX-TGCAAAGCCAACATTCCGCCAAATA-TAMRA-3 ', when probe and target sequence pairing, HEX fluorophor emitted fluorescence is because of approaching by cancellation with the TAMRA quencher of 3 ' end, when carrying out extension, 5 ' 5 prime excision enzyme activity of polysaccharase cuts off probe, make the HEX fluorophor separate emitting fluorescence with quencher TAMRA
Probe LSV-P is the sequence that 5 ' end 5-carboxyl-the X-rhodamine is modified, the dark quencher of 3 ' end is modified: 5 '-ROX-CCGTCGACTCCATCGCTGCG-ECLIPSE-3 ', when probe and target sequence pairing, ROX fluorophor emitted fluorescence is because of approaching by cancellation with the ECLIPSE quencher of 3 ' end, when carrying out extension, 5 ' 5 prime excision enzyme activity of polysaccharase cuts off probe, make FAM fluorophor and the dark essence agent ECLIPSE that goes out separate emitting fluorescence;
D, RT-Real time PCR product is carried out the real-time fluorescence positive detection;
E, according to the real-time fluorescence positive test symbol, judge whether the host is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus, positive expression host is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus, otherwise negative expression host is not infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Further, steps d also comprises the step that adopts blank that the RT-PCR reaction stability is detected; Reaction result is negative to illustrate that then RT-Real time PCR stable reaction is normal, and the positive RT-Real time PCR that then illustrates of reaction result reacts unstable, may be polluted, and needs to detect again.
Further, steps d also comprises the step that adopts negative control that RT-Real time PCR reaction stability is detected; Reaction result is negative to illustrate that then RT-Real time PCR stable reaction is normal, and the positive RT-Real time PCR that then illustrates of reaction result reacts unstable, may be polluted, and needs to detect again.
Further, in the described RT-Real time PCR reaction, the amplification cycles number of times is 40 times.
Further, in the b step, the RNA strength of solution is more than or equal to 10 of total rna concentration
-2Doubly.
Beneficial effect of the present invention: multiple RT-Real time PCR detection method provided by the invention can realize rapid detection that whether host (lily, gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus.In the RT-RealtimePCR system, add fluorophor, during the amplification of PCR index, measure the amount of specificity product immediately by the variation of continuous monitoring fluorescent signal power, and infer the original bulk of goal gene in view of the above, it is to be used for determining through the differential expression between the sample target transcript of different treatment or the target transcript differential expression in the different periods, rapidly and efficiently, and have very high susceptibility and a specificity.This multiple detection method can realize infecting the disposable synchronous detection of three kinds of viruses of lily and gladiolus, and trace routine is easy.
Embodiment
The present invention adopts the detection of multiple RT-Real time PCR detection technique realization to multiple virus.RNA according to arabis mosaic virus, bean virus 2 and lily asymptomatic virus, design specific amplimer and fluorescent probe, and carry out RT-Real time PCR reaction by the RNA template that amplimer and fluorescent probe are treated identifying virus, by detection, realize treating the rapid detection whether identifying virus comprises arabis mosaic virus, bean virus 2 and three kinds of viruses of lily asymptomatic virus to RT-Realtime PCR product.
The substance of technical solution of the present invention comprises: the multiple real-time RT-Real time PCR detection method of one, setting up arabis mosaic virus, bean virus 2 and lily asymptomatic virus; Two, specific amplimer and fluorescent probe have been designed;
Three, RT-Real time PCR stability monitoring.
One, the multiple RT-Real time PCR detection method of arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Reaction system: cumulative volume is 10 μ L reaction systems.10 * One Step RNA PCR Buffer, 1 μ L wherein, 25mmol/LMgCl
22 μ L, 10mmol/L dNTP Mixture 1 μ L, 40U/ μ L RNase Inhibitor 0.2 μ L, 5U AMVReverse Transcriptase XL 0.2 μ L, 5U AMV-Optimized Taq 0.2 μ L, the upstream primer of 10 μ mol/L ArMV, BYMV and LSV and downstream primer respectively are 0.4 μ L, 5 μ mol/L ArMV, BYMV and LSV probe respectively are 0.4 μ L, the RNA template of ArMV, BYMV and LSV respectively is 0.2 μ L, RNase Free dH
2O 1.2 μ L.
Response procedures: 50 ℃ of reverse transcription 30min; 95 ℃ of pre-sex change 10min; 95 ℃ of sex change 15s, 60 ℃ of annealing with extend 40s, 40 circulations.
Two, designed specific amplimer and fluorescent probe capsid protein gene sequence, design detection probes and primer according to ArMV, BYMV and LSV high conservative.
Arabis mosaic virus sequence length 65bp, its primer and fluorescent probe sequence are:
Upstream primer ArMV-F:5 '-TTTATGCGATCTCGGGACCTA-3 ';
Downstream primer ArMV-R:5 '-TTGCACAACCACTGGCATCT-3 ';
Probe ArMV-P:5 '-FAM-TGCCCCAAGTGGAGAAACTGCA-TAMRA-3 '.
BYMV sequence length 90bp, its primer and fluorescent probe sequence are:
Upstream primer BYMV-F:5 '-GGAGCAGGTGACATACCCTTTAA-3 ';
Downstream primer BYMV-R:5 '-TCCGCAACTTCCGAGAAATG-3 ';
Probe BYMV-P:5 '-HEX-TGCAAAGCCAACATTCCGCCAAATA-TAMRA-3 '.
LSV sequence length 72bp, its primer and probe sequence are:
Upstream primer LSV-F:5 '-GCAGCATTGAGTTTGAGAATGG-3 ';
Downstream primer LSV-R:5 '-GTCCAGCGTGCTTCTTCATG-3 ';
Probe LSV-P:5 '-ROX-CCGTCGACTCCATCGCTGCG-ECLIPSE-3 '.
Three, RT-Real time PCR stability monitoring
The total RNA that goes out with viral extracting to be detected is a template, and blank and negative control are set, and verifies the stability of multiple RT-Realtime PCR method.
Embodiment 1
The detection of adopting multiple RT-Real time PCR that whether host A (gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Host A (gladiolus) is detected, specifically comprise the steps:
Step a, extracting goes out total RNA from host A (gladiolus); Remove materials such as the polysaccharide that influences RT-Real time PCR reaction, phenols;
Step b, total RNA is diluted to 10
-2RNA solution doubly;
Step c, with RNA solution as template, carry out real-time RT-Real time PCR reaction;
Real time RT-PCR reaction system: cumulative volume is 10 μ L reaction systems.10 * One Step RNA PCR Buffer1 μ L wherein, 25mmol/L MgCl
22 μ L, 10mmol/L dNTP Mixture 1 μ L, 40U/ μ L RNase Inhibitor 0.2 μ L, 5 U AMV Reverse Transcriptase XL, 0.2 μ L, 5U AMV-Optimized Taq 0.2 μ L, upstream primer and the downstream primer of 10 μ mol/L primer ArMV, primer BYMV and primer LSV respectively are 0.4 μ L, 5 μ mol/L probe ArMV, probe BYMV and probe LSV respectively are 0.4 μ L, the RNA template of ArMV, BYMV and LSV respectively is 0.2 μ L, RNase Free dH
2O 1.2 μ L.
RT-Real timePCR response procedures: 50 ℃ of reverse transcription 30min; 95 ℃ of pre-sex change 10min; 95 ℃ of sex change 15s, 60 ℃ of annealing with extend 40s, 40 circulations.
Primer ArMV, primer BYMV and primer LSV are respectively according to the high conservative capsid protein gene sequences Design of arabis mosaic virus, bean virus 2 and lily asymptomatic virus, and three groups of amplimers not doing mutually between primer, three groups of amplimers have following sequence respectively:
Upstream primer ArMV-F:5 '-TTTATGCGATCTCGGGACCTA-3 ',
Downstream primer ArMV-R:5 '-TTGCACAACCACTGGCATCT-3 ',
Upstream primer BYMV-F:5 '-GGAGCAGGTGACATACCCTTTAA-3 ',
Downstream primer BYMV-R:5 '-TCCGCAACTTCCGAGAAATG-3 ',
Upstream primer LSV-F:5 '-GCAGCATTGAGTTTGAGAATGG-3 ',
Downstream primer LSV-R:5 '-GTCCAGCGTGCTTCTTCATG-3 ',
Probe ArMV-P, probe BYMV-P and probe LSV-P are respectively according to three kinds of fluorescent probes of the capsid protein gene sequences Design of arabis mosaic virus, bean virus 2 and lily asymptomatic virus high conservative, and it has following sequence respectively:
Probe ArMV-P:5 '-FAM-TGCCCCAAGTGGAGAAACTGCA-TAMRA-3 '
Probe BYMV-P:5 '-HEX-TGCAAAGCCAACATTCCGCCAAATA-TAMRA-3 '
Probe LSV-P:5 '-ROX-CCGTCGACTCCATCGCTGCG-ECLIPSE-3 ';
Steps d, in real time the RT-PCR product is carried out fluorescence positive detection, Detection of Stability;
Step e, judge whether host A (gladiolus) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.Blank, negative control show that all demonstration is negative, and the positive result of positive control shows RT-Real time PCR stable reaction, and detected result can adopt.The testing sample detected result shows positive, so host A (gladiolus) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Comparative Examples 1
The detection of adopting the DAS-ELISA detection method that whether host A (gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Adopt DAS-ELISA detection method, branch respectively host A (gladiolus) to be carried out arabis mosaic virus, bean virus 2 and lily asymptomatic virus three times and infect detection, final detection result is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus for host A (gladiolus) simultaneously, detected result is identical with embodiment 1 detected result, but DAS-ELISA detection method very complicated.
Embodiment 2
The detection of adopting multiple RT-Real time PCR that whether host B (oriental hybrid lily) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
This detection step and embodiment 1 detection method are basic identical, and difference is the host's difference among the step a.
Finally, blank, negative control show that all demonstration is negative, and positive control is positive, and the testing sample detected result shows positive, the result shows that this host B (oriental hybrid lily) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Comparative Examples 2
Adopt the DAS-ELISA method to detect the detection whether host B (oriental hybrid lily) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Adopt DAS-ELISA method, branch respectively host B (oriental hybrid lily) to be carried out arabis mosaic virus, bean virus 2 and lily asymptomatic virus three times and infect detection, final detection result is infected by lily asymptomatic virus for host B (oriental hybrid lily), and detected result is identical with embodiment 2 detected results.
Embodiment 3
The detection of adopting multiple RT-Real time PCR that whether host C (Asia lily) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
This detection step and embodiment 1 detection method are basic identical, and difference is the host's difference among the step a.
Finally, blank, negative control show that all demonstration is negative, and positive control is positive, and the testing sample detected result is positive, and the result shows that this host C (Asia lily) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Comparative Examples 3
Adopt the DAS-ELISA method to detect, host C (Asia lily) is carried out arabis mosaic virus, bean virus 2 and lily asymptomatic virus infect detection
Adopt the DAS-ELISA method, divide the detection of respectively whether host C (Asia lily) being infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus for three times, final detection result is that host C (Asia lily) is infected by lily asymptomatic virus, detected result is identical with embodiment 3 detected results, but DAS-ELISA method very complicated.
The detection that embodiment 4 adopts multiple RT-Real time PCR that whether host D (oriental hybrid lily) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
This detection step and embodiment 1 detection method are basic identical, and difference is the host's difference among the step a.
Finally, blank, negative control show that all demonstration is negative, and positive control is positive, and the testing sample detected result shows positive, the result shows that this host D (oriental hybrid lily) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Comparative Examples 4
Adopt the DAS-ELISA method to detect the detection whether host D (oriental hybrid lily) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Adopt DAS-ELISA method, branch respectively host D (oriental hybrid lily) to be carried out arabis mosaic virus, bean virus 2 and lily asymptomatic virus three times and infect detection, final detection result is infected by arabis mosaic virus for host D (oriental hybrid lily), detected result is identical with embodiment 4 detected results, but DAS-ELISA method very complicated.
Embodiment 5
The detection of adopting multiple RT-Real time PCR that whether host E (gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
This detection step and embodiment 1 detection method are basic identical, and difference is the host's difference among the step a.
Finally, blank, negative control show that all demonstration is negative, and positive control is positive, and the testing sample detected result is positive, and the result shows that this host E (gladiolus) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Comparative Examples 5
Adopt the DAS-ELISA method to detect the detection whether host E (gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Adopt DAS-ELISA method, branch respectively host E (gladiolus) to be carried out arabis mosaic virus, bean virus 2 and lily asymptomatic virus three times and infect detection, final detection result is infected by bean virus 2 for host E (gladiolus), detected result is identical with embodiment 4 detected results, but DAS-ELISA method very complicated.
Embodiment 6
The detection of adopting multiple Real time RT-PCR that whether host F (gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
This detection step and embodiment 1 detection method are basic identical, and difference is: total RNA extracts from host F (gladiolus), known viruse ToRSV, known viruse TRSV, known viruse LMoV and known viruse CMV among the step a.
Finally, blank, negative control show that all demonstration is negative, positive control is positive, the testing sample detected result shows positive, the result shows, the RNA template comprises viral arabis mosaic virus, bean virus 2 and lily asymptomatic virus, and promptly this host F (gladiolus) is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
Comparative Examples 6
Adopt the DAS-ELISA method to detect the detection whether host F (gladiolus) is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus
Adopt DAS-ELISA method, branch respectively host F (gladiolus) to be carried out arabis mosaic virus, bean virus 2 and lily asymptomatic virus three times and infect detection, final detection result is infected by arabis mosaic virus, bean virus 2 and lily asymptomatic virus for host F (gladiolus), detected result is identical with embodiment 5 detected results, show that detection method of the present invention has specificity, virus ToRSV, TRSV, LMoV and CMV do not have influence to monitoring result, the detection sensitivity height.
Claims (8)
1. whether arabis mosaic virus, bean virus 2 and lily asymptomatic virus infect host's real-time fluorescent RT-PCR method for detecting, it is characterized in that comprising the steps:
A, go up total RNA that extracting goes out virus to be detected from the host;
B, total RNA is diluted to RNA solution;
C, with RNA solution as template, carry out RT-Real time PCR reaction; In RT-Real time PCR reaction system, comprise amplimer, fluorescent probe; Wherein amplimer is totally three groups, is respectively primer ArMV, primer BYMV and primer LSV, has following sequence respectively:
Upstream primer ArMV-F:5 '-TTTATGCGATCTCGGGACCTA-3 ',
Downstream primer ArMV-R:5 '-TTGCACAACCACTGGCATCT-3 ',
Upstream primer BYMV-F:5 '-GGAGCAGGTGACATACCCTTTAA-3 ',
Downstream primer BYMV-R:5 '-TCCGCAACTTCCGAGAAATG-3 ',
Upstream primer LSV-F:5 '-GCAGCATTGAGTTTGAGAATGG-3 ',
Downstream primer LSV-R:5 '-GTCCAGCGTGCTTCTTCATG-3 ',
Primer ArMV, primer BYMV and primer LSV be respectively according to the high conservative capsid protein gene sequences Design of arabis mosaic virus, bean virus 2 and lily asymptomatic virus, and do not do mutually between primer,
Wherein fluorescent probe is totally three kinds, is respectively probe ArMV-P, probe BYMV-P and probe LSV-P; Wherein
Probe ArMV-P is the sequence that 5 ' end 5-Fluoresceincarboxylic acid is modified, 3 ' end 5-carboxyl tetramethyl-rhodamine is modified:
5′-FAM-TGCCCCAAGTGGAGAAACTGCA-TAMRA-3′，
Probe BYMV-P is the sequence that 5 ' end chlordene-6-methyl fluorescein is modified, 3 ' end 5-carboxyl tetramethyl-rhodamine is modified:
5′-HEX-TGCAAAGCCAACATTCCGCCAAATA-TAMRA-3′，
Probe LSV-P is 5 ' sequence that agent that end 5-carboxyl-the X-rhodamine is modified, the dark essence of 3 ' end is gone out is modified:
5′-ROX-CCGTCGACTCCATCGCTGCG-ECLIPSE-3′，
Probe ArMV-P, probe BYMV-P and probe LSV-P are respectively according to the capsid protein gene sequences Design of arabis mosaic virus, bean virus 2 and lily asymptomatic virus high conservative;
D, in real time RT-Real time PCR product is carried out the fluorescence positive detection;
E, according to the fluorescence positive test symbol, judge whether the host is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus, positive expression host is infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus, otherwise negative expression host is not infected by arabis mosaic virus, bean virus 2 or lily asymptomatic virus.
2. whether arabis mosaic virus according to claim 1, bean virus 2 and lily asymptomatic virus infect host's real-time fluorescent RT-PCR method for detecting, it is characterized in that steps d comprises the step that adopts blank that RT-Real time PCR reaction stability is detected; Reaction result is negative to illustrate that then RT-Real time PCR stable reaction is normal, and the positive RT-Real time PCR that then illustrates of reaction result reacts unstable, may be polluted, and needs to detect again.
3. whether arabis mosaic virus according to claim 1, bean virus 2 and lily asymptomatic virus infect host's real-time fluorescent RT-PCR method for detecting, it is characterized in that steps d also comprises the step that adopts negative control that RT-Real time PCR reaction stability is detected; Reaction result is negative to illustrate that then RT-Real time PCR stable reaction is normal, and the positive RT-Real time PCR that then illustrates of reaction result reacts unstable, may be polluted, and needs to detect again.
4. whether arabis mosaic virus according to claim 1, bean virus 2 and lily asymptomatic virus infect host's real-time fluorescent RT-PCR method for detecting, it is characterized in that the amplification cycles number of times is 40 times in the described RT-Real time PCR reaction.
5. whether arabis mosaic virus according to claim 1, bean virus 2 and lily asymptomatic virus infect host's real-time fluorescent RT-PCR method for detecting, it is characterized in that RNA strength of solution in the b step is more than or equal to 10 of total rna concentration
-2Doubly.
6. the amplimer of arabis mosaic virus is characterized in that, upstream primer and downstream primer have following sequence respectively:
Upstream primer: 5 '-TTTATGCGATCTCGGGACCTA-3 ',
Downstream primer: 5 '-TTGCACAACCACTGGCATCT-3 '.
7. the amplimer of bean virus 2 is characterized in that, upstream primer and downstream primer have following sequence respectively:
Upstream primer: 5 '-GGAGCAGGTGACATACCCTTTAA-3 ',
Downstream primer: 5 '-TCCGCAACTTCCGAGAAATG-3 '.
8. the amplimer of lily asymptomatic virus is characterized in that, upstream primer and downstream primer have following sequence respectively:
Upstream primer: 5 '-GCAGCATTGAGTTTGAGAATGG-3 ',
Downstream primer: 5 '-GTCCAGCGTGCTTCTTCATG-3 '.
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| CN112877354A (en) * | 2021-01-29 | 2021-06-01 | 中国林业科学研究院林业新技术研究所 | Method for obtaining intermediate genome sequence of poplar mosaic virus VIGS vector and application thereof |
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